Flow Cytometry (Direct immunofluorescence staining):
1. Prepare single-cell suspensions from either lymphoid tissue, bone marrow, peripheral blood or cell cultures using standard protocols.
2. Wash the cells twice in cold Stain Buffer (FBS) and pellet the cells by centrifugation (e.g., 300 x g at 4°C). Resuspend the cell pellet with cold Stain Buffer (FBS) to a final concentration of 2 x 10e7 cells/ml.
3. Distribute 50 µl aliquots of the cell suspension (10e6 cells) to either tubes or the round-bottomed wells of microwell plates.
4. Dilute fluorescent antibodies to their predetermined optimal concentrations in Stain Buffer (FBS) and add small aliquots (e.g., 10 µl) of the diluted antibodies to the tubes or microwells that contain the target cell suspensions. Incubate for 20 minutes on ice protected from light. Staining time may be increased (≥ 45 min) depending on the avidity of the fluorescent antibody.
5. Wash the cells two times with either 200 µl (for microwell plates) or 1 ml (for tubes) volumes of Stain Buffer (FBS) to remove unbound antibodies. Centrifuge cells as 300 x g for 5 min. After each centrifugation, carefully aspirate (for microwell plates or tubes) or invert and blot away (for tubes) supernatants from cell pellets.
6. Resuspend the cell pellet in either 200 µl (for microwell plates) or 0.5 ml (for tubes) volumes of Stain Buffer (FBS). Transfer stained cells from microwell plates to the appropriate tubes for flow cytometric analysis (adjust final volume to ~0.5 ml).
7. Analyze stained cell samples by flow cytometry as soon as possible (e.g., ≤ 4 hours) after staining. If analysis must be delayed, then the stained cells can be fixed with buffered paraformaldehyde (e.g., BD Cytofix™ Fixation Buffer; Cat. No. 554655) and stored at 4°C (protected from light). The fixed cells should be analyzed as soon as possible (e.g., up to one week after staining and fixation).
Note 1: Stain Buffer (FBS) can similarly be used for the indirect immunofluorescent staining of cells. In this case, repeat steps 4 and 5 when using either unlabeled or biotinylated primary antibodies. When staining cells with biotinylated antibodies and fluorochrome-conjugated avidin, it is desirable to use a staining medium that contains no biotin, such as Stain Buffer (BSA) (Cat. No. 554657).
Note 2: Stain Buffer (FBS) can also be used for the immunofluorescent staining of surface antigens expressed by cells that are destined to be fixed and immunofluorescently stained for intracellular antigens such as cytokines. Cells stained for intracellular cytokines can be resuspended and maintained (i.e., at 4°C, protected from light) in Stain Buffer (FBS) prior to analysis by flow cytometry.